Suboptimal trophectoderm mitochondrial DNA level is associated with delayed blastocyst development



To provide a comprehensive analysis of mtDNA quantity in D5 and D6 blastocysts, as well as a further insight to the origin of delayed blastocyst development.


A retrospective cohort analysis of 829 D5 and 472 D6 blastocysts from 460 patients who underwent in vitro fertilization (IVF) with next-generation sequencing (NGS)–based preimplantation genetic testing for aneuploidy (PGT-A). The quantity of trophectoderm mtDNA was extrapolated from the NGS data, followed by the analysis of mean mtDNA levels between D5 and D6 blastocysts of the same ploidy (aneuploid/euploid) and transfer outcomes (positive/negative clinical pregnancy).


D5 blastocysts had significantly higher euploidy rate and clinical pregnancy rate when compared with D6 blastocysts. The proportion of blastocysts derived from patients ≧ 40 years old were similar between the D5 and D6 cohorts. When blastocysts with identical ploidy were analyzed, the D5 cohorts all had significantly higher mean mtDNA levels than their D6 counterparts. Similarly, when embryo transfers with identical outcome were analyzed, the D5 cohorts also had significantly higher mean mtDNA levels than the D6 cohorts. Trophectoderm mtDNA level was independent of maternal age and blastocyst morphology grades.


Our data provided further evidence D5 blastocysts contained significantly greater mtDNA quantity than D6 blastocysts, and mtDNA quantity could be a key factor that affects the development rate of blastocysts. Furthermore, one must avoid using an arbitrary threshold when incorporating mtDNA quantity into the embryo selection criteria, as the observed value may have vastly different clinical implication when blastulation rate is also considered.

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Data availability

All data and material are available upon request.


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The authors would like to thank all of the patients who participated in the present study, as well as the IVF laboratory team members at IHMED Fertility center for their assistance.

Author information




Study design and coordination: Frank Shao-Ying Wu and Ni-Chung Lee

Subject recruitment: Shao-Peng Weng, Meng-Shun Shen, and Pei-Chun Ma

Data collection: Shao-Peng Weng, Meng-Shun Shen, and Po-Kuan Wu

Data analysis: Frank Shao-Ying Wu, Ni-Chung Lee, Po-Kuan Wu, and Pei-Chun Ma

Drafting of manuscript: Frank Shao-Ying Wu

Study supervision and manuscript revision: Ni-Chung Lee

Corresponding author

Correspondence to Ni-Chung Lee.

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The study protocol was approved by the institutional review board of the National Taiwan University Hospital (IRB #201905053RIND).

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Wu, F.SY., Weng, SP., Shen, MS. et al. Suboptimal trophectoderm mitochondrial DNA level is associated with delayed blastocyst development. J Assist Reprod Genet 38, 587–594 (2021).

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  • Mitochondria DNA quantity
  • Day 5 blastocysts
  • Day 6 blastocysts
  • IVF
  • Blastulation rate